It is our objective to identify as large a number of peptides and individual mRNA species that are "senescence-specific" as possible. By senescence-specific we mean peptides and mRNA species that are present in different amounts in senescent and presenescent (phase III and phase II) cultured human fibroblasts. Two screening assays will be used. The first will employ two-dimensional polyacrylamide gel electrophoresis and will screen for differences in synthesis, posttranslational modification and degradation of the abundant cellular peptides. The second will employ the replica blot procedure to compare hybridization of cDNA made from polysomal polyadenylated RNA of senescent cells to that of presenescent cells against cloned human genomic DNA aattached to the vector Charon 4A. Any differences will be further analyzed as follows. If the difference is due to differences in degradation or posttranslational modification then antibody will be made against the peptide in question and used to more precisely identify the rate of degradation or the nature of the posttranslational modification. If the difference is due to a difference in the amount of functional mRNA in the polysomes then individual cloned fragments of human genomic DNA complementary to the specific senescence-specific mRNA will be isolated. These cloned DNA fragments will then be used as probes to measure rates of synthesis, nuclear processing, nuclear and cytoplasmic degradation and efficiency of translation of the cytoplasmic mRNA. In all of these studies, when the cause of a difference is determined, an attempt will be made to determine and characterize the enzymatic basis for this difference. For senescence-specific peptides or mRNA species, a correlation will be made with the onset of senescence and the induction/repression of the peptide or mRNA species. We propose by this approach to begin a comprehensive study of the changes in gene activities that accompany the onset of senescence. This approach will reveal basic control points in cell physiology as well as begin to elucidate the basic mechanisms operating during cellular aging.